Antimutagenic compositions for treatment and prevention of photodamage to skin

ABSTRACT

A method of improving DNA repair and reducing DNA damage and for reducing mutation frequency in skin for the purpose of reducing consequences of exposure to solar or ultraviolet radiation is disclosed. The methods comprise administering to the skin a composition containing deoxyribonucleosides in concentrations sufficient to enhance DNA repair or reduce mutation frequency in a vehicle capable of delivering effective amounts of deoxyribonucleosides to the necessary skin cells.

REFERENCES TO RELATED APPLICATIONS

This application is a continuation in part of U.S. application Ser. No.08/963,831, filed Nov. 4, 1997, herein incorporated by reference.

FIELD OF THE INVENTION

This invention relates generally to treatment and prevention ofphotodamage, genetic damage, and tumorigenesis in skin and other tissuescaused by exposure to solar or ultraviolet radiation or other mutagens,comprising administration of deoxyribonucleosides or esters ofdeoxyribonucleosides to a mammal such as a human. These compounds arecapable of reducing DNA damage, mutation frequency, and tumorigenesiswhen applied topically before, during, or after exposure to mutagenicradiation or chemical mutagens.

BACKGROUND OF THE INVENTION

Exposure of skin to ultraviolet (or ionizing) radiation damages DNA,which if unrepaired or improperly repaired, can lead to carcinogenesisas well as contribute to acceleration of the aging process. DNA damageand consequent genomic instability are defining characteristics of bothcarcinogenesis and biological aging. Patients with defective DNA repaircapabilities in diseases like xeroderma pigmentosa display prematureskin aging and a very high incidence of skin cancers (Robbins andMoshell, J. Inv. Dermatol., 73:102-107, 1979) on sun-exposed areas ofthe skin. Pharmacological intervention in damage to skin due to solar orultraviolet radiation has heretofore been largely restricted to agentslike sunscreens or free-radical scavengers intended to prevent damage,or agents like retinoic acid or glycolic acid which are intended toremodel the surface of radiation-damaged skin without necessarilyaddressing the most fundamental mechanisms of cell or tissue damage andrepair at the level of genomic integrity.

In practice, preventive measures like sunscreen use are not completelyeffective, and exposure to sunlight is not always anticipated. Theincidence of skin cancers in the United States approaches 1,000,000cases per year. Therefore, there exists a need for a therapeutic agentwhich will reduce the risk of development of skin cancer or otherconsequences of skin photodamage even when applied after exposure tosunlight has already occurred. Sunscreens and agents which induce orimprove tanning are not useful in such situations, since they are onlyuseful if applied prior to exposure to UV radiation. Moreover, there aresituations wherein sunscreens and even endogenous melanin can actuallyenhance UV-induced DNA damage through photodynamic sensitization.

There have been several attempts to improve or accelerate DNA repair andto reduce the consequences of DNA damage in skin cells after damage hasalready occurred. The first major step in DNA repair is detection andexcision of damaged portions of DNA. The viral enzyme T4 endonuclease Vcan accomplish this step with some forms of DNA damage. T4 endonucleaseV, when packaged in epidermis-penetrating liposomes, has been shown toaccelerate the rate of excision of pyrimidine dimers, the most commonform of photolesion, in photodamaged skin of mice in vivo (Yarosh etal., Cancer Res. 52:4227-31, 1992). A bacterial extract has beenreported to increase the rate of unscheduled DNA synthesis, which isoften used as an index of DNA repair activity (Kludas and Heise, U.S.Pat. No. 4,464,362), in UV-exposed skin; however, this effect was notconfirmed in a subsequent study (Natarajan et al., Mutation Research206:47-54, 1988).

The key issue in DNA repair, however, is not necessarily the rate oflesion excision, but the fidelity of repair. Agents which accelerate theexcision step of DNA repair can actually exacerbate damage if the cellsare incapable of accurate repair synthesis at a rate that matches therate of excision of damaged segments of DNA (Collins and Johnson, J.Cell Physiol. 99:125-137, 1979).

Deoxyribonucleosides or deoxyribonucleotides have been added to cells inculture with variable or divergent effects on DNA damage or mutagenesisin response to irradiation of the cells. In some cell types, e.g.lymphocytes, which have limited capabilities for de novodeoxyribonucleotide synthesis, exogenous deoxynucleosides are reportedto improve cell survival after exposure to UV radiation (Yew andJohnson, Biochim. Biophys. Acta, 562:240-251, 1979; Green et al.,Mutation Research, 350:239-246, 1996) or ionizing radiation (Petrovic etal., Int. J. Radiat. Biol., 18:243, 1970); no significant improvement insurvival was seen after addition of deoxyribonucleosides toUV-irradiated normal human fibroblasts (Green et al., Mutation Research,350:239-246, 1996). A crucial point is that increasing cell survivalafter genomic damage caused by UV radiation or other mutagens is notnecessarily desirable. The process of programmed cell death, orapoptosis, is integrated with cellular mechanisms for detecting DNAdamage. Thus, genomic damage which by itself is not sufficient to causecell death, can trigger apoptosis, an active cellular suicide process,so that the DNA damage in the cell is not perpetuated in subsequent cellgenerations, with tumorigenesis as a possible outcome as genomic damageaccumulates. The mechanisms for detecting genomic damage and inducingapoptosis involve cell-cycle regulating proteins such as thetumor-suppressor protein p53. Therefore, agents which promote cellsurvival (e.g. by inhibiting apoptosis) after irradiation are notnecessarily anticarcinogenic, and may actually enhance mutationfrequency and risk of malignant transformation by permitting survival ofdamaged cells that would otherwise be eliminated by apoptosis. Asignificant illustration of this principle is the demonstration thatembryonic p53 knockout mice exposed to ionizing radiation in utero havea higher survival rate (live birth) than wild-type controls, but alsohave a much higher frequency of congenital defects (Norimura et al.,Nature Medicine, 2:577-580).

In studies where the effect of exogenous deoxyribonucleosides onmutation frequency in UV-irradiated cells has been explicitly studied,variable results have been obtained. Bianchi and Celotti (MutationResearch 146:277-284, 1985) reported that thymidine or deoxycytidine athigh concentrations increased the mutation frequency in UV-irradiatedV79 Chinese hamster cells; no reduction in mutation frequency wasobserved at any concentrations of added nucleosides. Musk et al.(Mutation Research 227:25-30, 1989) reported that a mixture ofdeoxyribonucleosides which included excess deoxycytidine relative to theother nucleosides, reduced the mutation frequency in response to UV-C(254 nm) irradiation in MM96L melanoma cells, a cell line with a knownconstitutive excess of purine deoxyribonucleotides. In the same study,exogenous deoxyribonucleosides had no effect on mutation frequency inanother neoplastic cell line, human HeLa cells, after exposure to UV-Cradiation. It is important to note that UV-C radiation is not acomponent of solar radiation at the surface of the earth, since it isblocked effectively by the atmosphere (Pathak, 1974, in Sunlight andMan, ed. by T. B. Fitzpatrick, University of Tokyo Press, Tokyo, Japan,p. 815). The effect of deoxyribonucleosides on mutation frequency incells exposed to solar radiation or UV radiation at wavelengths that arepresent in solar radiation was not tested, and the authors explicitlyconclude their discussion with the statement “ . . . the lower[mutation] frequency in sun-[irradiated] compared with UVC-irradiatedMM96L cells suggests that sunlight either does not perturb thedeoxynucleoside pools or it induces a cellular response that isinsensitive to nucleoside levels.”

In addition to agents which inhibit apoptosis or improve DNA repairsufficiently to permit cell survival but not necessarily for correctionof potentially tumorigenic mutations, growth factors in general(including those that are involved in normal wound healing responseslike TGF-β or PDGF) act as tumor promoters.

U.S. Pat. No. 5,246,708 discloses the methods and compositions involvingthe use of mixtures of deoxyribonucleosides for promotion of the healingof wounds, ulcers, and burns, including those caused by ultraviolet orsolar radiation.

Acyl derivatives of deoxyribonucleosides have been taught as deliverymolecules for promoting entry of deoxyribonucleosides into the skin, asdisclosed in U.S. patent application Ser. No. 08/466,379. It isdisclosed that acyl derivatives of deoxyribonucleosides can improvecellular repair and cell survival after damage to skin caused byradiation.

Oligodeoxyribonucleotides have been proposed as melanogenic stimuli,based on the idea that DNA damage, or excision products of DNA damage,might be cellular signals for increasing melanin production in the skinto help protect against subsequent damage. Gilchrest et al. (U.S. Pat.No. 5,470,577; WO Application Serial No. 95/01773) proposed thatexogenous DNA photodamage products may stimulate melanogenesis withoutactual damage to cellular DNA as a necessary intermediate step. Thestated intention was to mimic the presence of cyclobutane pyrimidinedimers or other DNA photodamage products in order to provide the cellwith false DNA damage signals that might trigger induction ofmelanogenesis in the absence of actual DNA damage. Treatment of melanomacells in vitro and guinea pig skin in vivo with thymidine dinucleotideresulted in increases in melanin production. The authors stated thatthey believed that DNA fragments entered the cells, and even theirnuclei, intact. They proposed that sunless tanning accomplished over aperiod of weeks by topical administration of oligodeoxyribonucleotides,especially thymidine dinucleotide, could protect skin by inducingmelanin synthesis, with consequent reduction of passage of UV radiationinto and through the skin.

Wiskemann (1974; in Sunlight and Man, ed. by T. B. Fizpatrick,University of Tokyo Press, Tokyo, Japan, p. 51) reported that systemic(intraperitoneal) administration the deoxyribonucleoside thymidine orthe ribonucleosides and congeners adenosine, cyclic-AMP, uridine,cytidine increased the period of latency for extravasation ofsystemically administered Evan's Blue dye in the skin in the first fewhours after UV exposure, indicating a reduction in acute UV-inducededema. In this system, DNA administered after irradiation had no effecton extravasation of dye. The author also explicitly states thatnucleobases incorporated into ointments do not penetrate the horny layer(the stratum corneum, the outer layer of enucleated keratinocytescomprising the main moisture barrier of skin) of human epidermis.

OBJECTS OF THE INVENTION

It is an object of the invention to provide compositions and methods forreducing mutation frequency, photaging, and tumorigenesis in skin,thereby attenuating consequences of exposure to solar and ultravioletradiation and to other mutagens including endogenous oxidants.

It is an object of the invention to provide a composition that enhancesDNA repair and prevents consequences of mutagenic radiation even whenadministered after damage or exposure to radiation or other mutagens hasalready occurred.

It is a primary object of this invention to provide compositions andmethods for effectively preventing or treating consequences of exposureof the skin to solar and ultraviolet radiation and other environmentalmutagens.

It is a further object of the invention to provide compositions andmethods for improving the activity of chemical sunscreens.

It is a further object of the invention to provide compositions andmethods for reducing deleterious effects of sunscreens and othercompounds, exogenous and endogenous, which act as photosensitizing orphotodynamic enhancers of UV-induced damage to skin.

It is a further object of the invention to provide compositions andmethods for reducing some consequences of inflammatory skin and mucosalconditions, including psoriasis, dermatitis and inflammatory boweldisease.

SUMMARY OF THE INVENTION

The subject invention involves methods and compositions for improvingDNA repair (or genomic fidelity) and reducing photodamage in skinexposed to ultraviolet radiation, ionizing radiation, or chemicalmutagens by topical administration of compositions containing effectiveamounts of a source of deoxyribonucleosides. The compositions arecapable of delivering deoxyribonucleosides to the necessary targetcells. Such compositions are applied to the skin before, during or afterexposure to solar, ultraviolet, or ionizing radiation, or chemicalmutagens including but not limited to endogenous or exogenous sources offree radicals or nitric oxide. Treatment with compositions of theinvention improves the net fidelity of DNA repair and thereby reducesmutation frequency and the risk of tumorigenesis in response to solar orultraviolet radiation or other mutagens.

The invention provides methods and compositions for deliveringdeoxyribonucleosides to skin cells in concentrations sufficient tosupport and improve repair of damaged DNA and to reduce deleteriousconsequences of exposure of skin to radiation or chemical mutagens.

In addition to prevention of consequences of exposure to sunlight,compounds and compositions of the invention are useful for treating skinlesions caused by sunlight like actinic keratoses or solar lentigenes.

The deoxyribonucleosides are administered either as freedeoxyribonucleosides, or as derivatives thereof which are converted todeoxyribonucleosides after application to the skin. Such derivativesinclude deoxyribonucleotides, oligonucleotides, DNA itself, and acylderivatives of deoxyribonucleosides or other derivatives ofdeoxyribonucleosides which are converted to free deoxyribonucleosides byendogenous enzymes.

Methods and compositions of the invention also improve activity andreduce side effects of other agents used on skin for prophylactic,therapeutic, or cosmetic purposes, including but not limited tosunscreens, retinoids, alpha-hydroxy acids, methylxanthines, and DNArepair enzymes.

The invention also relates to compositions and methods for reducingdeleterious consequences (e.g. cellular damage, especially to DNA, whichcan result in increased likelihood of mutations or other potentiallycarcinogenic damage to the genome) of endogenous and exogenousphotochemically-active compounds or chromophores which act asphotosensitizers or photodynamic enhancers of DNA damage caused by solaror ultraviolet radiation.

The invention, as well as other objects, features and advantages thereofwill be understood more clearly and fully from the following detaileddescription, when read with reference to the accompanying results of theexperiments discussed in the examples below.

DETAILED DESCRIPTION OF THE INVENTION

DNA damage and repair is involved in the development of skin cancer andphotoaging. The subject invention provides compounds which successfullyimprove the net fidelity of DNA repair. The subject invention will haveimportant consequences in health care and will improve the cosmeticappearance of skin.

A. Definitions

The term “deoxyribonucleoside” refers to any one of the four principlenucleoside constituents of DNA: deoxyadenosine, deoxycytidine,deoxyguanosine, and thymidine. The term “ribonucleoside” refers to anyone of the four major nucleoside constituents of RNA: adenosine,cytidine, guanosine, and uridine.

The term “acyl derivative of a deoxyribonucleoside” refers todeoxyribonucleosides bearing acyl substituents derived from carboxylicacids (which modify the pharmacokinetics and bioavailability of the freedeoxyribonucleosides), as disclosed in U.S. patent application Ser. No.08/466,379, hereby incorporated by reference in its entirety.

The term “source of at least one deoxyribonucleoside” or“deoxyribonucleoside source” in the context of the subject inventionrefers to deoxyribonucleosides themselves or derivatives ofdeoxyribonucleosides which can be converted to deoxyribonucleosides byendogenous enzymes, especially esterases. Examples include acylderivatives of deoxyribonucleosides (carboxylic acid esters),deoxyribonucleotides (phosphate esters), or oligodeoxyribonucleotides(phosphate diesters). Since esterase activity (involving various enzymescapable of cleaving carboxylic acid esters and phosphate esters) isubiquitous in mammalian tissues including skin, these esters ofdeoxyribonucleosides are converted to deoxyribonucleosides when appliedto skin. Similarly, a “source of at least one ribonucleoside” refers toa ribonucleoside or ribonucleoside ester, including a ribonucleotide, anoligoribonucleotide, or an acyl derivative of a ribonucleoside.

The term “ester of a deoxyribonucleoside” (or deoxyribonucleoside ester)refers to either an acyl derivative of deoxyribonucleosides as describedabove or to a phosphate ester of a deoxyribonucleoside (ordeoxyribonucleosides), e.g. deoxyribonucleotides,oligodeoxyribonucleotides, or polydeoxyribonucleotides.

The term “photosensitization” in the context of the subject inventionrefers to the process whereby light-absorbing (UV or visible light)molecules directly transfer the energy of an excited state, generally atriplet state, to a target molecule, resulting in damage to DNA andother cellular structures. The target molecule can be DNA itself oranother target which results in damage to DNA, e.g. membranes componentsof lysosomes, which contain deoxyribonuclease.

The term “photodynamic sensitization” herein refers to the processwhereby UV-absorbing molecules generate free radical species or otherdiffusible reactive intermediates as a result of excitation by UV orvisible radiation.

The term “sunscreen agents” refers to a UV-absorbing chemicals that areintended to be used in sunscreen products as active ingredients forreducing exposure of the skin to the UV component of solar radiation.Examples of sunscreen agents currently used as such in commercialproducts include avobenzone (t-butyl dimethoxydibenzoylmethane),oxybenzone (benzophenone-3), dioxybenzone (benzophenone-8),sulisobenzone (benzophenone-4;2-hydroxy-4-methoxybenzophenone-5-sulfonic acid), octocrylene(2-ethylhexyl-2-cyano-3,3-diphenylacrylate), octyl methoxycinnamate(2-ethylhexyl p-methoxycinnamate), octyl salicylate(2-ethylhexylsalicylate), homosalate (homomenthyl salicylate), trolaminesalicylate (triethanolamine salicylate), phenylbenzimidazole sulfonicacid, PABA (para-aminobenzoic acid), roxadimate (ethyl 4-bishydroxypropyl aminobenzoate), lisadimate (glyceryl PABA), Padimate O(octyldimethyl PABA), menthyl anthranilate, and Parsol 1789 (butylmethoxydibenzoylmethane).

The term “energy scavenger” refers to a compound which absorbs energyfrom the excited states of sunscreen agents or endogenousphotosensitizing or photodynamic enhancers of DNA damage, reducingdamage to target biological molecules like DNA. In this context, energyscavengers should be nontoxic at effective concentrations and shouldyield a net reduction in damage to DNA when present during UVirradiation in the presence of a photosensitizer or photodynamicenhancer of DNA damage. In the context of this invention, “energyscavengers” refer particularly to compounds with lowest triplet stateswith energies less than or equal to those of the nucleobase constituentsof genomic DNA. The primary quality of energy scavengers of theinvention is that, by virtue of photochemical energy state properties,or mass action (concentration), they prevent damage to genomic DNA thatwould otherwise occur due to energy transfer for excited chromophores,whether endogenous (e.g. melanin) or exogenous (e.g. sunscreens). Energyscavengers with photochemical properties similar to those of structuralconstituents of DNA are advantageous, and include sources of at leastone deoxyribonculeoside or ribonucleoside, like a deoxyribonucleoside,an acyl deoxyribonucleoside, a deoxyribonucleotide, anoligodeoxyribonucleotide, a ribonucleoside, a ribonucleotide, anoligoribonucleotide, and an acyl ribonucleoside.

The term “deleterious consequences” as used herein refers to cellulardamage in a mammal caused by a mutagen, especially damage to the genome,resulting in an increased chance of developing skin cancer or other skinlesions like solar lentigines, actinic keratoses, or other signs ofphotoaging like skin wrinkles or “age spots”. Mutagens capable ofcausing such deleterious consequences include solar radiation,ultraviolet radiation, ionizing radiation, free radicals (whetherproduced as a result of irradiation of a photochemically activechromophore or from some other source, including normal metabolicprocesses), nitric oxide, and environmental mutagens.

B. Compounds of the Invention

The compounds of the invention are primarily the majordeoxyribonucleoside constituents of DNA: deoxyadenosine, deoxycytidine,deoxyguanosine, and thymidine. The invention also includes the use ofeffective amounts of precursors of these deoxyribonucleosides, e.g.oligodeoxyribonucleotides, DNA, deoxyribonucleotides and acylderivatives of deoxyribonucleosides, and, particularly for minimizationof effects of photodynamic sensitizers and photosensitizing agents onDNA, ribonucleosides and their congeners, e.g. oligoribonucleotides,ribonucleotides, and acyl derivatives of ribonucleosides.

2-Deoxyribose and acyl derivatives of 2-deoxyribose are also usefulcompounds of the invention. They are particularly advantageous for usein treatment of existing sunlight-induced skin lesions like actinickeratoses.

While not wishing to be bound by a theory, it is believed that theactive agents of the invention that pass into cells are thedeoxyribonucleosides or acyl derivatives of deoxyribonucleosides, sincethe anionic phosphate moiety on deoxyribonucleotides oroligodeoxyribonucleotides impedes passage across cell membranes.Phosphorylated deoxyribonucleoside precursors are converted to freedeoxyribonucleosides by enzymatic and nonenzymatic degradation before orafter application to the skin, prior to their entry into cells.

The deoxyribonucleosides are produced by any of several methods. Theyare produced by degradation of DNA from biological sources, e.g. fishsperm, by chemical synthesis, or by fermentation technology.

Also encompassed by the invention are pharmaceutically acceptable saltsof the above-noted compounds.

C. Compositions of the Invention

The invention includes pharmaceutical compositions for improving the netfidelity of DNA repair and for protecting the skin against mutagens. Thecomposition comprises 1) an effective amount of a source of one or moredeoxyribonucleosides, and optionally 2) an effective amount of apharmaceutically acceptable topical carrier capable of delivering thedeoxyribonucleosides or their precursors to appropriate target cells inthe skin under in vivo conditions.

While individual deoxyribonucleosides, especially deoxycytidine (seeExample 7) have some activity in attenuating UV-induced tumorigenesis,two or more deoxyribonucleosides, or preferably all four, are typicallyincluded in a formulation of the invention. Encompassed by the inventionare compositions containing deoxyadenosine, deoxycytidine,deoxyguanosine, or thymidine, either as single agents, or in allpossible combinations of two, three, or all four of these compounds.Compositions containing deoxycytidine are particularly advantageous. Theconcentrations of individual deoxyribonucleosides in compositionsencompassed by the invention, whether present individually or incombination with other deoxyribonucleosides or deoxyribonucleosideprecursors (such as deoxyribonucleoside esters), and normalized to theamount of free nucleoside or nucleoside moiety in the case ofdeoxyribonucleoside phosphates or oligonucleotides or prodrugs likeacylated deoxyribonucleoside derivatives, range from 0.1 to 10 mg/ml,advantageously 1 to 5 mg/ml.

In the case of deoxyribose or acyl derivatives of deoxyribose,appropriate concentrations in a composition of the invention range from0.1 to 100 millimolar, advantageously 10 to 50 millimolar.

In order to permit access of the deoxyribonucleosides and relatedcompounds of the invention to deeper-lying skin cells, vehicles whichimprove their penetration through the outer layers of the skin, e.g. thestratum corneum, are useful. Vehicle constituents which improve thepenetration of compounds of the invention into the skin include but arenot limited to: ethanol, isopropanol, diethylene glycol ethers such asdiethylene glycol monoethyl ether, azone(1-dodecylazacycloheptan-2-one), oleic acid, linoleic acid, propyleneglycol, hypertonic concentrations of glycerol, lactic acid, glycolicacid, citric acid, and malic acid.

Appropriate concentrations of diethylene glycol monoehtyl ether incompositions of the invention range from 2 to 20 percent, advantageouslyfrom 5 to 15 percent, on a weight/weight basis.

In addition to promoting absorption of agents into the skin, use oftopical alpha-hydroxy acids (AHA), e.g. lactic acid and glycolic acid,can affect the ability of the skin to reduce the penetration ofultraviolet light into the vulnerable basal layers of the epidermis.Thus, there is also an increased need for agents which reduce theconsequences of exposure to solar or ultraviolet radiation in peopleusing AHA's, e.g. for promoting exfoliation of epidermal cells. Sincepenetration of UV-absorbing sunscreen agents into the skin isundesirable because of possible photosensitization and photodynamicenhancement of UV-induced damage to cells, the compounds of theinvention are uniquely suitable for combination with AHA's, either inthe same formulation or a separate one, for improving skin resistance todamaging effects of solar or ultraviolet radiation.

One embodiment of the invention is a hydrogel formulation, comprising anaqueous or aqueous-alcoholic medium and a gelling agent, and adeoxyribonucleoside source. Suitable gelling agents include but are notlimited to methylcellulose, carboxymethylcellulose,hydroxypropylmethylcellulose, carbomer (carbopol), hypan, polyacrylate,and glycerol polyacrylate.

Concentrations of gelling agents are selected according to their effecton viscosity and pharmaceutical and cosmetic propoerties. Suitableconcentrations of a carbomer gelling agent, e.g. carbomer 934P, rangefrom 1 to 15%, advantageously 2 to 10% on a weight/weight basis.

Liposomes are microscopic lipid vesicles which can containpharmacologically active agents either enclosed in the aqueous spacewithin the vesicle or in the lipid membrane itself, depending on thelipophilicity of the agent. Liposomes are capable of delivering apharmacologic agent through the stratum corneum into deeper layers ofthe skin, and are therefore suitable vehicles for compounds andcompositions of the invention.

Niosomes are lipid vesicles similar to liposomes with membranesconsisting largely of non-ionic lipids, some forms of which areeffective for transporting compounds across the stratum corneum.

In one embodiment of the invention, lipophilic acyl derivatives ofdeoxyribonucleosides, e.g. oleic or palmitic acid esters ofdeoxyribonucleosides are incorporated into membranes of niosome orliposome membranes, in addition to or instead of being enclosed withinthe vesicular membranes.

Other agents which are advantageous for incorporation into a compositionof the invention include corticosteroids, especially hydrocortisone inconcentrations of 0.05 to 1%, other anti-inflammatory corticosteroids attherapeutically effective concentrations, topical anesthetics includingbut not limited to benzocaine, lidocaine, and benzyl alcohol, aloe veraand aloe barbadensis, retinoids, antioxidants like Vitamins C and E,flavins, polyphenols (e.g. extracted from green tea or black tea),allantoin, liposomal DNA repair enzymes, antibacterial agents (e.g.quaternary ammonium compounds, bacitracin, neomycin, polymyxin), zincsalts, and methylxanthines. All of these listed agents have some utilityin treating or attenuating various aspects of skin injury or discomfortcaused by ultraviolet radiation or inflammatory skin conditions, and aretherefore complementary to the unique actions of thedeoxyribonucleosides of the invention.

Benzyl alcohol, which is known to have anesthetic and preservativeproperties, has the unexpected effect of improving aqueous solubility ofthe relatively insoluble purine deoxyribonucleosides, deoxyadenosine anddeoxyguanosine; preferred concentrations of benzyl alcohol in topicalformulations of deoxyribonucleosides are 0.5 to 5%. This is veryimportant in permitting high concentrations of the deoxyribonucleosidesof the invention to be stably incorporated into aqueous vehicles.

Sunscreens

The compounds of the invention are advantageously incorporated into thesame formulation as a UV-absorbing chemical sunscreen agent such as:avobenzone (t-butyl dimethoxydibenzoylmethane), oxybenzone(benzophenone-3), dioxybenzone (benzophenone-8), sulisobenzone(benzophenone-4; 2-hydroxy-4-methoxybenzophenone-5-sulfonic acid),octocrylene (2-ethylhexyl-2-cyano-3,3-diphenylacrylate), octylmethoxycinnamate (2-ethylhexyl p-methoxycinnamate), octylsalicylate(2-ethylhexylsalicylate), homosalate (homomenthyl salicylate),trolamine salicylate (triethanolamine salicylate), phenylbenzimidazolesulfonic acid, PABA (para-aminobenzoic acid), roxadimate (ethyl 4-bishydroxypropyl aminobenzoate), lisadimate (glyceryl PABA), Padimate O(octyldimethyl PABA), menthyl anthranilate, or Parsol 1789 (butylmethoxydibenzoylmethane).

Alternatively, the compounds of the invention are formulated in a basewhich is suitable for application to the skin prior to, or after,application of a sunscreen. This embodiment of the invention permits thebenefits of deoxyribonucleosides, in reducing consequences of UV damageand in attenuating photodynamic enhancement of cellular damage caused bysunscreen agents themselves, to be obtained in conjunction with use of awide variety of commercial sunscreen products.

D. Therapeutic Uses of the Compounds and Compositions of the Invention

Reduction of Deleterious Consequences of Exposure of Skin to Ultravioletor Solar Radiation

The compounds and composition of the invention, when applied oradministered before, during, or after exposure of the skin of a mammalto mutagenic radiation, have the unexpected activity of improving thenet fidelity DNA repair, thereby reducing mutation frequency,photoaging, and the chance of developing skin cancers.

DNA repair proceeds by several steps. A chemical lesion in DNA, whichcan be caused by ultraviolet radiation, ionizing radiation, freeradicals, or chemical mutagens, is detected by the DNA repair system. Asegment of nucleotides including the damaged region is excised,generally along with a number of surrounding nucleotides. The excisedstrand is then resynthesized from free deoxyribonucleotides, using theintact DNA strand as a template.

It is generally believed that improvement of repair of DNA in skin cells(e.g. keratinocytes, melanocytes, fibroblasts) would require alterationin the activity of enzymes or other proteins involved in the detectionand excision of DNA lesions. Thus, there have been attempts to improveDNA repair in skin by delivering DNA repair enzymes viatopically-applied liposomes (U.S. patents (Yarosh et al., Cancer Res.52:4227-31, 1992).

Far more important than the initial rate of excision of lesions is thefidelity or accuracy of repair. Agents which accelerate the excisionstep of DNA repair can actually exacerbate damage if the cells areincapable of accurate repair synthesis at a rate that matches the rateof excision of damaged segments of DNA (Collins and Johnson, J. CellPhysiol. 99:125-137, 1979).

Compounds and compositions of the invention, when applied to skinbefore, during or after exposure to solar or ultraviolet radiation orother environmental mutagens, reduces the mutation frequency otherwiseinduced by the mutagen (Example 1). This reduces the damage to cellscaused by the mutagen, and reduces the chance of development of skincancers, as shown in Examples 2 and 7. The effect on genomic fidelity isin principle mediated by actual improvement in the fidelity of repair inan individual cell or by improvement of the elimination of cells withirreparable DNA damage. Either mechanism results in a net improvement ingenomic fidelity in skin exposed to mutagens.

The compounds of the invention have unanticipated benefits when used incombination with other agents known to be useful in various aspects ofskin care, including but not limited to sunscreens, methylxanthines,retinoids, DNA repair enzymes, exfoliants, and protease inhibitors,corticosteroids and nonsteroidal anti-inflammatory agents.

The compounds and compositions of the invention, when applied soonenough, e.g. within about 3 days after exposure to ultraviolet or solarradiation, improve the repair of cellular and macromolecular damage andimprove net genomic fidelity, thereby reducing the chance of developmentand severity of macroscopically visible deleterious consequences of suchexposure, including but not limited to photoaging, sunburn symptoms,actinic keratoses, solar lentigines, “age spots”, and skin cancer, e.g.basal cell carcinoma, squamous cell carcinoma, melanoma. The compoundsand compositions of the invention are also optionally applied before orduring exposure to solar or ultraviolet radiation to shorten the timegap between damage and onset of repair enhancement by the compounds ofthe invention.

Treatment of skin with compounds and compositions of the inventionresults in a reduced chance of development of skin cancers and otherdeleterious consequences of exposure to solar or UV radiation likephotaging even when the compounds of the invention are applied evenafter irradiation, e.g. after unintended exposure topotentially-damaging doses of solar radiation. This type of activity isnot shared by conventional sunscreens or agents which might act byenhancing melanogenesis, which are useful only if applied beforeirradiation.

Compounds and compositions of the invention are furthermore useful fortreating existing inflammatory or hyperproliferative skin lesions,especially those caused by exposure to sunlight or ultravioletradiation, including but not limited to actinic keratoses, solarlentigines, and wrinkles. Example 9 illustrates this activity in apatient, in which topical application of a composition of the inventionresulted in complete regression of an an existing actinic keratosis.

Improvement of Sunscreen Activity and Attenuation of PhotodynamicEnhancement of UV Damage by Sunscreens

Sunscreens are typically designed and tested on the basis of preventionof sunlight-induced erythema. While erythema and its attenuation bysunscreens is an important short-term effect, reduction of erythema andinflammation by sunscreens does not necessarily mean that they produce aproportionate protection of DNA (or prevention of skin cancers and somefeatures of photoaging secondary to DNA damage). Sunscreens arecertainly useful in preventing some manifestations of photoaging andUV-related carcinogenesis, but do not provide complete protection, andin some situations may actually exacerbate photoinjury by acting asphotodynamic sensitizers (see Example 3).

Chemical sunscreens are intended to act by absorbing photons atmutagenic (or erythmogenic) wavelengths, thus producing a short-livedexcited singlet state; return to the ground state is accompanied byphoton emission at longer wavelengths that are supposed to be lessharmful than the incident radiation. Photon emission during the rapidreturn of a molecule from an excited singlet state to a ground state isknown as “fluorescence”. However, sunscreens or other exogenous orendogenous UV-absorbing molecules can also be excited to longer-livedtriplet states which can facilitate further reactions (theenergy-emission that occurs during return of a molecule from an excitedtriplet state to a ground state is known as “phosphorescence”, andtypically occurs over a much longer time span than fluorescence). Theconsequence is that some UV-absorbing agents, especially those with alowest triplet state that has a higher energy level than the lowesttriplet state of genomic DNA constituents, can absorb photons andactually exacerbate damage to DNA by direct or indirect energy transfer(e.g. from a triplet excited state) rather than by simple fluorescence,or photon emission at harmless wavelengths.

Benzophenone, a close structural analog of oxybenzone, increases theyield of strand breaks and pyrimidine dimers in UV-irradiated DNA, andis known to produce free radicals upon irradiation with UV light(Charlier et al., Photochemistry and Photobiology, 15:527-536, 1972).The results presented in Examples 3, 4, 5 and 6 indicate that a similarphenomenon also occurs with approved sunscreen ingredients, and that thedeoxyribonucleosides of the invention attenuate this deleteriousconsequence of sunscreen use.

When present in concentrations sufficient to block access of UVradiation to cells, sunscreen agents are protective. However, if presentin very low concentrations, insufficient to adequately block UVtransmission, photon-absorbing agents, including common sunscreeningredients, can operate as energy-transfer molecules, efficientlytrapping UV energy and transferring it to cell components, eitherdirectly (photosensitization) or by catalyzing formation of reactiveoxygen radicals (photodynamic sensitization). Thus, low concentrationsof oxybenzone, for example, enhance DNA damage induced by ultravioletradiation, whereas higher concentrations, sufficient to block UV accessto the cells or their immediate microenvironment altogether, protectagainst DNA damage (see Example 3).

The expected activity in vivo is that an oxybenzone-containing sunscreenwould protect cells from damage if present in a layer sufficient toblock access of light to the target cells altogether. However, at lowerconcentrations, insufficient to prevent penetration of UV radiation totarget cells, and especially if some oxybenzone has been absorbed intothe critical cell layers, either via passage through the stratum corneumor through hair follicles, there may be potentiation of damage to DNA invivo. In mice treated topically with commercial sunscreen containingoxybenzone, effects consistent with this hypothesis are in fact observedafter exposure to UV (see Example 6). Classes of sunscreens other thanbenzophenone derivatives also exacerbate UV-induced damage to DNA whenpresent at low concentrations during irradiation.

A strain of transgenic mice (v-HA-ras transgenic TG.AC mice) which isvery susceptible to a variety of carcinogens, including UV radiation hasbeen developed recently (Leder et al., Proc. Nat. Acad. Sci. USA,87:9178-9182, 1990). In response to a relatively small exposure to UVradiation, these mice reliably develop cutaneous papillomas within a fewweeks. When a circular patch of commercial sunscreen is applied to theback of such a mouse, the center of the protected region does in facthave lower incidence of UV-induced papillomas than the unprotected side,but often, along the margin of the applied sunscreen, there is a veryhigh incidence (sometimes higher than in unprotected areas) ofpapillomas (See Example 6). A layer of sunscreen sufficient to block UVaccess to target cells is protective, but low concentrations, e.g. atthe margin of a patch of sunscreen) can act as photosensitizersincreasing the incidence of a UV-induced skin cancer beyond that seen incompletely “unprotected” skin. Exposure of relevant skin cells to lowphotosensitizing (rather than protective) concentrations of sunscreensclearly must occur during ordinary usage, e.g. at the margin of anapplied patch, or as a protective layer is washed or worn off.

A benefit of deoxyribonucleosides or related compounds added toconventional sunscreen formulations (or other cosmetics containingsunscreens), beyond the support of DNA repair, is to synergize withconventional sunscreen compounds by acting as energy scavengers whichtrap energy emitted by (or radicals produced by) sunscreen agents thatis chemically similar to the cellular target, DNA. Exogenousdeoxyribonucleosides (in addition to their direct absorbance of UVenergy) serve as “decoys” for energy captured by sunscreen agents orother photosensitizers that would otherwise be transferred to cellulartargets, including DNA. Thus, deoxyribonucleosides provide adose-dependent reduction in damage caused to cellular DNA by UVradiation in the presence of low concentrations of photosensitizingagents like oxybenzone or other sunscreen agents (Example 4).

A defining characteristic of suitable energy scavenging agents is thattheir lowest triplet energy state is equal to or lower than that of DNAconstituents in situ. Because of the similarity of physicochemicalproperties of deoxyribonucleosides (or deoxyribonucleotides, acyldeoxyribonucleosides, or oligodeoxyribonucleotides) and genomic DNA, thecompounds of the invention are particularly suitable asenergy-scavenging agents to protect genomic DNA from damage due toenergy transfer from photosensitizers or photodynamic sensitizers. Inthis embodiment, ribonucleosides, ribonucleotides, oligoribonucleotidesand acyl derivatives of ribonucleosides are within the scope of theinvention. Appropriate concentrations of such scavengers in acomposition for topical application range from 0.1 to 100 milligrams permilliliter (normalized to the amount of free nucleoside present in thecase of nucleotides or oligonucleotides or acyl derivatives ofnucleosides). Advantageously, such scavengers are present inconcentrations ranging from 0.1 to 20 mg/ml or especially 1 to 5 mg/ml.

The problem of photodynamic enhancement of damage to DNA extends beyondsunscreens. Other compounds including endogenous molecules in the skin,can absorb UV radiation at wavelengths that do not necessarily directlydamage DNA significantly, and transfer that energy to cellular targetsincluding DNA, or generate free radicals that damage cellular DNA.Examples of endogenous photosensitizing or photodynamically active skinconstituents (photochemically active chromophores) include but are notlimited to porphyrins, tryptophan, riboflavin, and melanin. Exogenousphotodynamically active compounds include psoralens, which are presentin some perfume oils (bergamot), and which are in fact used to enhancesunlight-induced tanning and UV phototherapy of psoriasis throughexacerbation of cellular injury. Many therapeutic drugs or theirmetabolites are photochemically active chromophores which can produceskin adverse reactions when a patient is exposed to solar, visible, orultraviolet radiation. Pigments and other light-absorbing constituentsof cosmetics are also photochemically active chromophores which canexacerbate cellular photodamage.

The deoxribonucleosides and related compounds of the invention (e.g.deoxyribonucleotides, oligonucleotides, or DNA itself) are useful forattenuating cellular damage caused by excited light-absorbing molecules,including exogenous photochemically active chromophores like sunscreensand cosmetic pigments, and also from endogenous chromophores liketryptophan, porphyrins, urocanic acid and melanin.

Furthermore, since the photodynamic enhancement of DNA damage caused bybenzophenone derivatives is in part mediated by production of freeradicals (Charlier et al., Photochemistry and Photobiology, 15:527-536,1972), compounds and compositions of the invention are useful forprotecting the skin and mucosa from free radical damage, whether or notthe free radicals (e.g. hydroxyl radicals, peroxide radicals, orlipoperoxide radicals) are initiated or produced by photodynamicphenomena. Examples 3,4,5 and 6 provide evidence that thedeoxyribonucleosides of the invention protect against DNA caused by freeradicals.

The deoxyribonucleosides and related compounds of the invention areadvantageously incorporated into the same formulation as chemicalsunscreen agents, which include but are not limited to: avobenzone(t-butyl dimethoxydibenzoylmethane), oxybenzone (benzophenone-3),dioxybenzone (benzophenone-8), sulisobenzone (benzophenone-4),octocrylene (2-ethylhexyl-2-cyano-3,3-diphenylacrylate), octylmethoxycinnamate (2-ethylhexyl p-methoxycinnamate), octyl salicylate(2-ethylhexylsalicylate), homosalate (homomenthyl salicylate), trolaminesalicylate (triethanolamine salicylate), phenylbenzimidazole sulfonicacid, PABA (para-aminobenzoic acid), roxadimate (ethyl 4-bishydroxypropyl aminobenzoate), lisadimate (glyceryl PABA), Padimate O(octyldimethyl PABA), menthyl anthranilate, or Parsol 1789 (butylmethoxydibenzoylmethane). Such sunscreen agents are present informulations at concentrations that are in accord with regulatoryguidelines and standard use.

Similarly, in another embodiment of the invention, compounds of theinvention are incorporated into cosmetics containing photochemicallyactive chromophores to minimize deleterious consequences of combinedexposure of skin to solar or ultraviolet radiation and such cosmeticingredients.

Alternatively, the compounds of the invention are applied to the skin ina separate composition, e.g. a spray, lotion, roll-on, stick, or gel,before or after a sunscreen product or cosmetic is applied.

Methylxanthines

Methylxanthines, such as caffeine, theophylline, aminophylline orisobutylmethylxanthine, have been proposed as “sunless” tanning agents,which act by modulating activity of the biochemical pathways involved inmelanogenesis. Their proposed mechanism involves inhibition ofcyclic-AMP phosphodiesterase, thus enhancing the biological activity ofcyclic AMP in the pathways regulating melanogenesis. Methylxanthines canenhance the production of melanin in melanocytes, acting either alone orin combination with tanning stimulants like ultraviolet or solarradiation. However, methylxanthines are also known to exacerbate DNAdamage caused by ultraviolet radiation, which has been attributed to animpairment of DNA repair or disruption of cell cycle control mechanisms(Kastan et al., Cancer Research, 51:6304-6311, 1991).

Compounds of the invention are useful for reducing the deleteriouseffect of methylxanthines on DNA damage caused by exposure of skin toultraviolet or solar radiation. The compounds of the invention thusimprove the safety of skin tanning products that contain methylxanthinesas active ingredients. Compounds of the invention are applied eitherseparately or in the same formulation as the methylxanthines.

Exfoliants

Cosmetics containing alpha-hydroxy acids (AHA) such as lactic acid,glycolic acid, citric acid, or malic acid are widely used. They havemoisturizing and exfoliant properties. Products containing highconcentrations of AHA are also used to produce more extreme “skinpeels”, in which the outer layers of the epidermis are removed,essentially by means of a chemical burn. New epidermis growing in isoften softer and smoother than the skin layers that were removed.Beta-hydroxy acids like salicylic acid are also useful exfoliants.Retinoic acid is used for similar purposes through its exfoliant actionsand through stimulation of epidermal cell turnover and alteration ofepidermal metabolism.

Exfoliants are reported to reduce the sun-blocking capabilities of thestratum corneum, and furthermore increase the permeability of the skinto other agents. Thus, there is a need for use of UV protection inconjunction with AHA's, yet the increased skin permeability produced byAHA requires caution in the selection of sun protection agents, sinceabsorbed sunscreen agents can produce photodynamic enhancement of DNAdamage. The compounds of the invention are effective in reducingdeleterious consequences of exposure to sunlight or ultravioletradiation or other environmental mutagens in subjects using exfoliants,including but not limited to alpha-hydroxy acids, beta-hydroxy acids,and retinoids.

Nonsteroidal Anti-inflammatory Agents

Nonsteroidal anti-inflammatory agents are commonly used for treatment ofarthritis and other anti-inflammatory agents. Moreover, some members ofthis class, e.g. diclofenac (2,6-dichloro-phenyl-amino-phenylacetate)are under investigation as topical agents for reducing some aspects ofskin photodamage.

One of the prototypical members of this class of drugs, acetaminophen,inhibits DNA repair after damage caused by UV radiation by inhibitingthe enzyme ribonucleotide reductase, which converts ribonucleosidediphosphates to deoxyribonucleoside diphosphates (Hongslo et al.,Mutagenesis, 8:423-429, 1993). By supplying deoxyribonucleosides toskin, especially to areas of the skin that are generally exposed tosunlight, of patients receiving either oral or topical treatment withnonsteroidal anti-inflammatory agents, compositions of the inventionovercome a deleterious consequence associated with this widely-usedclass of drugs.

Ornithine Decarboxylase Inhibitors

One consequence of UV irradiation of skin is an increase in cellproliferation rate and in the activity of enzymes necessary for cellproliferation, one of which is ornithine decarboxylase (ODC).Difluoromethylornithine (DFMO) is an inhibitor of ODC, which isnecessary for polyamine synthesis, which in turn is necessary for DNAreplication. DFMO is useful for reducing the incidence of skin cancerand precancerous actinic lesions. Inhibition of cell cycling afterexposure to solar or UV radiation may give cells more time to repair DNAbefore mutagenic lesions are fixed by cell division, but cell cyclestasis also generally results in reduced levels of deoxyribonucleosides,which are necessary for repair of DNA damage. Deoxyribonucleosides aretherefore useful in conjunction with DFMO and other antiproliferativeagents which act via mechanisms not directly involving induced depletionor imbalance of deoxyribonucleotide pools. Deoxyribonucleosides of theinvention are optionally incorporated into the same formulation as DFMO(or other inhibitors of skin cell proliferation that act throughmechanisms other than impairment of DNA precursor synthesis), or areapplied in a separate formulation.

Treatment of Skin During Exposure to Endogenous Nitric Oxide

Nitric oxide (NO) is a biologically active mediator released byendothelial cells, macrophages and other cell types, especially duringinflammatory episodes. Nitric oxide is also an important mediator oferythema associated with UV exposure. Inhibitors of NO synthetaseattenuate the increase in skin blood flow following exposure(Deliconstantinos et al., J Cardiovasc Pharmacol 20 Suppl 12: S63-5,1992; Deliconstantinos et al., Br J Pharmacol 114(6): 1257-65, 1995;Warren, FASEB Journal, 8(2): 247-51, 1995).

Nitric oxide is a potent inhibitor of the enzyme ribonucleotidereductase (RR), which is the key enzyme for de novo synthesis ofdeoxyribonucleotides (Kwon et al., J Exp Med 174(4): 761-7, 1991;Lepoivre et al., J Biol Chem 269(34): 21891-7, 1994) Theantiproliferative effects of nitric oxide are in part attributable toinhibition of ribonucleotide reductase. This can be beneficial in aninflammatory response to an infectious microorganism or a neoplasm, butis deleterious in cells in need of capability for DNA repair, e.g. skinexposed to inflammatory mediators elicited by UV exposure. The amountsof NO released from macrophages are sufficient to inhibit ribonucleotidereductase and thereby induce cytostasis in neighboring cells.

The best-known inhibitor of RR, hydroxyurea (HU), has structuralsimilarities to N-omega-hydroxy-1-arginine, a physiological intermediatein NO production. Hydroxyurea can act as an NO-like nitrosating reactant(LePoivre et al., J Biol Chem 269(34): 21891-7, 1994). Both NO andhydroxyurea inhibit RR by quenching a tyrosyl radical in the active siteof the enzyme.

A discovery first disclosed herein is that NO sensitizes cells toUV-induced DNA damage via mechanisms that are reversible with exogenousdeoxyribonucleosides (see Example 8). Moreover, NO by itself, in theabsence of UV exposure, causes DNA damage that is prevented or reversedby compounds of the invention (Example 8).

The inflammatory response to UV exposure (which also plays an importantrole in UV-associated immunosuppression), via inhibition ofribonucleotide reductase by NO, exacerbates the mutagenic and cytotoxiceffects of UV exposure. The subject invention provides a means forcompensating for this deleterious effect of NO. Exogenousdeoxyribonucleosides, which enter into DNA metabolism downstream ofribonucleotide reductase, compensate for reduced RR activity. NO,although deleterious in cells needing deoxyribonucleotides, has somebeneficial effects in sun-exposed skin. Inhibitors of NO production alsoimpair the healing of skin damaged by exposure to excessive UVradiation, i.e., UV doses that produce a sufficiently severe sunburn forwound healing processes to be called into action (Benrath et al.,Neurosci Lett, 1995). Thus, NO is useful for tissue repair (probably byimproving blood flow and perhaps also by stimulating growth factorrelease from macrophages or keratinocytes), but may simultaneouslyexacerbate UV-related damage to DNA by reducing cellular capacities forproduction of deoxyribonucleotides for DNA repair.

NO-mediated inhibition of RR provides a mechanism for a disproportionateincrease in DNA damage without a corresponding increase in othersymptoms of sun exposure during repeated exposure to strong sunlight.Topical application of the compounds and compositions of the inventionprovides a method for ameliorating this form of conditionalhypersensitivity.

NO participates in skin inflammatory reactions that do not necessarilyinvolve exposure to solar or ultraviolet radiation. NO, which isreleased from activated macrophages, is component of most inflammatoryreactions. The compounds and methods of the invention provide a means ofovercoming some deleterious effects of NO in inflammatory skinconditions, including but not limited to psoriasis, dermatitis, allergicdermatitis, contact dermatitis (e.g. reactions to poison ivy and poisonoak), eczema and acne. In these conditions, NO sensitizes some celltypes to UV-induced DNA damage by inhibiting deoxyribonucleotidesynthesis. Compounds and compositions of the invention ameliorate thisdeleterious consequence of combined UV exposure and inflammatory skinconditions. Since NO and other endogenous oxidants that cause DNA damageare present in inflammatory skin conditions like psoriasis or dermatitiseven in the absence of significant exposure to UV radiation, thedeoxyribonucleosides of the invention are useful for protecting geneticintegrity of skin cells in inflammatory conditions. Compounds of theinvention prevent or repair DNA damage caused by NO alone, withoutexposure to UV radiation (Example 8).

Ribonucleosides are also useful for treating inflammatory skin anmucosal conditions, in part by providing higher concentrations of theribonucleotide substrates for ribonucleoside reductase. Ribonucleosides(or ribonucleoside esters) in this context are used in the same waysthat deoxyribonucleosides, advantageously in topically-appliedcompositions, with ribonucleosides (or ribonucleoside esters)incorporated in concentrations ranging from 0.1 to 20 mg/ml,advantageously 1 to 5 mg/ml. Adenosine is particularly useful fortreatment of inflammatory conditions.

Inhibition of DNA precursor synthesis by hydroxyurea leads to enhancedactivity and leakage of hydrolytic lysosomal enzymes which participatein extracellular damage, e.g. in inflammatory skin conditions orphotodamage (Malec et al., Chem. Biol. Interact., 57:315-324, 1986). Thecompounds of the invention prevent this component of inflammatory tissueinjury, especially when such inhibition of DNA precursor synthesis ismediated by endogenously-produced NO, which is functionally similar tohydroxyurea.

A further contribution to genomic damage is that exposure of cells toultraviolet radiation results in the release of enzymes, includingdeoxyribonuclease, from lysosomes. Deoxyribonuclease II, which ispresent in lysosomes, is an endonuclease that can produce strand breaksin nuclear DNA following lysosome disruption. Leakage of enzymes fromlysosomes also occurs in inflammatory conditions in general. Thecompounds and compositions of the invention are useful for preservinggenomic integrity after chromosomal damage caused by deoxyribonucleasereleased from lysosomes in inflammatory skin conditions and afterexposure of skin to solar or ultraviolet radiation.

By limiting some of the deleterious consequences of skin inflammation,compounds and compositions of the invention are useful asanti-inflammatory agents, and are optionally administered (either asseparate compositions or, advantageously, in the same formulation) inconjunction with other topical or systemic anti-inflammatory agentsincluding but not limited to corticosteroids like hydrocortisone and itscongeners.

Similarly, compounds and compositions encompassed by the invention areuseful for treatment of mucosal inflammatory conditions, including butnot limited to inflammatory bowel disease, ulcerative colitis, orCrohn's disease, or mucositis anywhere in the gastrointestinal tract.The preferred mode of treatment is by topical administration, in thiscase via enema or suppository, for which purposes deoxyribonucleosidesor other compounds of the invention are incorporated into suitablevehicles.

Treatment of Skin and Mucosal Tissues Exposed to Ionizing Radiation

Patients receiving therapeutic treatment (e.g. for cancer) with ionizing(X-Ray or gamma) radiation can suffer damage to skin overlying aninternal tumor, leading to desquamation and poor healing. Compounds andcompositions of the invention are useful for treating damage to skin andmucosal surfaces caused by intentional or accidental exposure toionizing radiation. For treatment of skin, compositions of the inventionare applied topically before or after radiation treatment. For treatmentof mucosal surfaces, e.g. in the mouth, gastrointestinal tract, urethraor vagina, appropriate compositions of the invention are also appliedtopically. Suitable compositions for treatment of mucosal surfacesinclude gels, lotions, ointments, suppositories, orally-administeredcapsules, pills or dragees, or solutions.

E. Administration and Formulation of Compounds and Compositions of theInvention

Compounds of the invention are formulated in pharmaceutically acceptablevehicles that deliver the compounds to the necessary cell populations inskin at concentrations adequate to accomplish the objectives of reducingmutation frequency and chance of developing cancer.

Compositions of the invention are applied before, during, or afterexposure to sunlight or other mutagens. A lotion or hydrogel containingdeoxyribonucleosides (0.1 to 10 mg/ml, advantageously 1 to 5 mg/ml) isapplied to skin as a thin film. The composition should be applied withinabout 48 or 72 hours after exposure to damaging doses of sunlight orultraviolet radiation, in order to provide support for DNA repair priorto the first cell divisions after irradiation, although applicationbefore, during, or within 12 hours after exposure to intense sunlight isadvantageous. Compositions of the invention are also effective whenapplied before exposure to radiation or other mutagens, as long as thedeoxyribonucleosides so provided, or their anabolites, are available tocells in need of their beneficial effects at the time of exposure to amutagen. Advantageously, compositions of the invention are appliedwithin about 12 hours before exposure of the skin to a solar radiationor other mutagens.

Compositions of the invention are advantageously applied as a daily-useskin treatment, once to several times per day, especially on sun-exposedparts of the body, or sites of inflammatory skin conditions. Exposure tosolar radiation leading to skin photodamage and photoaging is generallya cumulative process, involving repeated exposure to sunlight, evendaily, over a period of years. In this context, use of the compounds andcompositions of the invention to prevent or treat photodamage to theskin involves treatment of existing lesions due to prior sun exposure,as well as prevention of, or attenuation of the severity of, damage dueto present and future exposure to sunlight or other mutagens.

By improving repair of molecular damage to DNA as it occurs or before itis permanently established in the genome by cell division, or bypreventing initial damage through energy scavenging, compositions of theinvention prevent or delay the manifestation of deleterious consequencesof radiation, free radicals, or chemical mutagens, such as grosslyvisible skin damage, photoaging, actinic keratoses, and skin cancer.Thus, compositions and methods of the invention reduce the rate ofappearance and the incidence of signs of skin photodamage, especiallywhen administered regularly, e.g. daily, or especially before, during,or after exposure to solar radiation.

Compositions of the invention are also useful for promoting regressionof established sunlight-induced and other inflammatory andhyperproliferative skin lesions, e.g. actinic keratoses, contactdermatitis, psoriasis, eczema, or acne, or skin cancers like melanoma,basal cell carcinoma, or squamous cell carcinoma. In such conditions,topical gels, creams, ointments or lotions are applied to the affectedareas once or twice per day as needed.

In one embodiment of the invention, a composition containingdeoxyribonucleosides is applied to skin prior to application of asunscreen, as an alternative to use of formulations containing bothconventional sunscreens and deoxyribonucleosides or related compounds ofthe invention.

For treatment of colon mucosal inflammation, e.g. inflammatory boweldisease, compositions of the invention are administered as a suppositoryor enema, approximately once per day according to clinical need. Volumesof 10 to 500 ml of a suitable enema composition containingdeoxyribonucleosides are suitable for treatment of inflammatory boweldisease, e.g. ulcerative colitis or Crohn's disease. For treatment ofmucositis in other parts of the gastrointestinal, such as the mouth,standard pharmaceutically acceptable vehicles for that route ofadministration are used, e.g. mouthwashes or adherent hydrocolloids.

F. Synthesis of the Compounds of the Invention

Deoxyribonucleosides, being constituents of DNA, are present in allliving organisms, and can therefore in principle be extracted from avariety of sources. In practice, the most convenient biological sourcesat present are fish milt, which contains a relatively high concentrationof DNA. Fish milt sacs are homogenized, and the DNA therein is partiallypurified and treated with deoxyribonucleases and phosphatases to degradeit to the level of nucleosides, which are then purified bychromatography and recrystallization.

Since mixtures of deoxyribonucleosides are used in some embodiments ofthe invention, purified deoxyribonucleosides are recombined inappropriate proportions. Alternatively, deoxyribonuclosides are notseparated from each other during purification from other fish miltcomponents (or other contaminants if the DNA is derived from otherbiological sources); appropropriate quantities of individualdeoxyribonucleosides are added to such a mixture, if necessary, toadjust the relative proportions of deoxyribonucleosides.

Deoxyribonucleosides can also be synthesized chemically from simplerprecursors.

Acyl derivatives of deoxyribonucleosides, as disclosed in U.S. patentapplication Ser. No. 466,379, are useful for 1) providing sustainedavailability of deoxyribonucleosides due to gradual deacylation bynonspecific esterases in the skin, and 2) improved penetration throughhydrophobic biological membranes or extracellular media, e.g. theintercellular lipids in the stratum corneum of the epidermis.

It will be obvious to the person skilled in the art that other methodsof synthesis may be used to prepare the compounds of the invention.

The following examples are illustrative, but not limiting of the methodsand compositions of the present invention. Other suitable modificationsand adaptations of a variety of conditions and parameters normallyencountered in clinical therapy which are obvious to those skilled inthe art are within the spirit and scope of this invention.

EXAMPLE 1

Post-irradiation Topical Deoxyribonucleosides Improve DNA Repair inMouse Skin After UVB Exposure

The incidence of mutations in skin in response to ultraviolet radiationwas determined using the “Big Blue” transgenic mouse test system. Thesemice carry approximately 40 copies per cell of a lambda phage shuttlevector containing a lacI gene as a target for mutagenesis, as well asthe lad promoter, the lac operator, and the αlacZ reporter gene.

Following exposure of the mice to ultraviolet radiation and treatmentwith compounds of the invention or vehicle, genomic DNA from skinsamples is extracted and the shuttle vectors are packaged in lambdavirus heads. The phage lambda viruses containing the shuttle vectors areplated on E.Coli with the color reagent X-Gal, which turns blue whenenzymatically altered by galactosidase, the product of the αlacZ gene.Viruses containing nonmutated lacI genes produce white plaques; mutationof the lacI gene results in blue plaques. The mutation frequency isdetermined by counting the relative numbers of white and blue plaques.

Stratagene BigBlue™ transgenic mice (n=7/group) were shaved and thenirradiated the next day with UVB radiation (85% of energy at 313 nm),1.25 kJ/m2.

Deoxyribonucleosides (“dNsides”; 4 mg/ml each of deoxyadenosine,deoxycytidine, deoxyguanosine, and thymidine) or vehicle (propyleneglycol) were applied topically 30 minutes after irradiation and againeach day for 5 days.

Mice were sacrificed on day 5 after irradiation. DNA was extracted fromdorsal (irradiated) and ventral (non-irradiated) skin, packaged intolambda phage and plated on E.Coli along with X-Gal. Colonies withmutations were blue. >200,000 colonies were counted in each group.

TABLE 1 Topical deoxyribonucleosides reduce mutation frequency inUV-irradiated skin Spontaneous mutation frequency (nonirradiated skin)Average  4.5 × 10⁻⁵ Mutation frequency in UVB-irradiated skin TotalIncrement due to UV Control 34.9 × 10⁻⁵ 30.4 × 10⁻⁵ dNsides  7.8 × 10⁻⁵ 3.3 × 10⁻⁵

As shown in Table 1, post-irradiation treatment with topicaldeoxyribonucleosides reduced the incidence of mutations caused by UV-Bradiation by a factor of nearly 10 in this experiment (30.4×10⁻⁵ versus3.3×10⁻⁵)

EXAMPLE 2

Post-irradiation Treatment with Topical Deoxyribonucleosides PreventsDevelopment of UV-induced Papillomas in v-Ha-ras Transgenic TG.AC Mice

Example 1 demonstrated that post-irradiation topical treatment canreduce the frequency of UV-induced mutations in a reporter gene in “BigBlue” transgenic mice, through support and improvement of DNA repairprocesses. One of the consequences of reduced mutation frequency inresponse to a carcinogen like UV radiation should be a reduction ofUV-induced tumorigenesis.

A strain of transgenic mice has been developed which is extremelysensitive to carcinogens. It permits rapid determination of carcinogenicpotential of various chemical agents and other treatments. Normal micerequire repeated exposure to ultraviolet radiation over a number ofweeks in order to reliably develop skin tumors. In contrast, v-Ha-rasTG.AC transgenic mice can develop tumors rapidly after a singleexposure, or small number of exposures to UV radiation.

In 9 mice exposed to UV-B radiation (0.3-1.25 kJ/m²×3 q2d) and treatedafter irradiation with vehicle (propylene glycol), a total of 35papillomas were found 4 weeks after exposure.

Among 7 mice exposed to the same doses of UV-B radiation and treatedwith deoxyribonucleosides (4 mg/ml of each major deoxyribonucleoside inpropylene glycol) after irradiation, only 1 papilloma was observed(Table 2).

TABLE 2 Topical deoxyribonucleosides reduce UV-induced tumorigenesisControl 3.89 tumors/mouse dNside-treated 0.14 tumors/mouse

The beneficial effect of deoxyribonucleosides in reducing UV-inducedtumorigenesis must be due to improvement of repair phenomena or cellularproofreading, or to inhibition of tumor promotion, and not to preventionof initial damage, since the deoxyribonucleosides were applied afterirradiation. This observation indicates that deoxyribonucleosidetreatment after (or presumably also during) exposure to UV-B radiationhas important inhibitory effects on tumorigenesis, as a consequence ofimproved maintenance of genomic fidelity.

This antitumorigenic effect of topical deoxyribonucleosides even whenapplied after irradiation is particularly surprising in view of thereported efficacy of deoxyribonucleosides in accelerating wound healing(U.S. Pat. No. 5,246,708), since other growth factors known toaccelerate wound healing, like platelet-derived growth factor (PDGF) ortransforming growth factor beta (TGF-β), act as tumor promoters.

The antitumorigenic effect of deoxyribonucleosides in this experiment isalso unexpected in view of the beneficial effect of deoxyribonucleosideson survival of cells in culture when the deoxyribonucleosides areapplied after exposure of the cells to ultraviolet or ionizingradiation. Prevention of apoptosis of damaged cells would be expected toincrease the likelihood of tumor development, as occurs in animals withdefective p53-related mechanisms.

EXAMPLE 3

Low Concentrations of Oxybenzone Exacerbate UV-induced Damage to DNA

Confluent human fibroblasts in T25 flasks were washed 3 times with HBSS(Hank's Balanced Salt Solution) and incubated with vehicle or withvarious concentrations of oxybenzone (OB) for 2 hours. Media wasaspirated and cells were covered with a 1 mm layer of HBSS andirradiated from above with UV-B (50 J/m²). The medium was aspirated andcells were incubated for three hours with 2 mM hydroxyurea. Medium wasagain aspirated, and cells were trypsinized with 0.25% trypsin/EDTA.Cells were centrifuged at 4° C., resuspended in 50 microliters of HBSSand incubated at room temperature with 200 microliters of 1N NaOH for 15minutes. DNA damage (single strand breaks) was assessed by alkalinesucrose gradient centrifugation. “Nucleoid position” in the sucrosegradient is proportional to the number of DNA single strand breaks.

UV irradiation without oxybenzone results in a three to four-foldincrease in the nucleoid position over baseline (Table 3). Oxybenzone atthe higher concentrations tested, 20 and 200 micromolar, protectedcellular DNA against damage from the UV irradiation, as the nucleoidposition for these groups is close to that of non-irradiated cells.However, cells exposed to 2 micromolar OB display substantially moredamage than cells irradiated with no OB at all; nucleoid position valuesare 10-fold greater than those of non-irradiated cells. Thus, oxybenzonestrongly enhances DNA damage when present at low concentrations, whereasit protects cells at higher concentrations. In practice, even underconditions of proper sunscreen use, there will always be areas of skinexposed to low, potentially deleterious concentrations of sunscreen,either at the edge of a patch of applied sunscreen, or as an appliedlayer wears off over the course of a day.

TABLE 3 Low concentrations of oxybenzone enhance UV-induced DNA damageGroup Nucleoid Postion (mm) No UV  3 UV 50 J/m² 11 UV 50 J/m² + 2 μM OB32 UV 50 J/m² + 20 μM OB  4 UV 50 J/m² + 200 μM OB  3

EXAMPLE 4

Deoxribonucleosides Attenuate Photodynamic Enhancement of DNA DamageCaused by Oxybenzone

Confluent human fibroblasts were exposed to 2 micromolar oxybenzone(OB), as in Example 3, prior to exposure to UV-B radiation (50 J/m²).Different flasks of cells also were exposed to increasing concentrationsof deoxyribonucleosides. Cells were processed for determination ofnucleoid position in a sucrose density gradient, a measure of DNA singlestrand breaks.

As shown in Table 4, deoxyribonucleosides produce a dose-dependentreduction in the yield of DNA single strand breaks induced by UVexposure plus 2 μM OB. Deoxyribonucleosides at 2 μM slightly reduce DNAdamage; at 200 micromolar, the deoxyribonucleosides almost completelyabrogate the DNA damage.

TABLE 4 Deoxyribonucleosides attenuate photodynamic enhancement of DNAdamage caused by UV plus oxybenzone Group Nucleoid Postion (mm) UV + 2μM OB 56 UV + 2 μM OB + 2 μM dNsides 47 UV + 2 μM OB + 20 μM dNsides 23UV + 2 μM OB + 100 μM dNsides 22 UV + 2 μM OB + 200 μM dNsides  7

EXAMPLE 5

Effect of Individual Versus Combined Deoxribonucleosides onPhotodynamically-enhanced, UV-induced DNA Damage

Human fibroblasts were prepared and treated as in Example 4, and theeffects of individual deoxyribonucleosides on photodynamically enhancedDNA damage (2 μM oxybenzone) were determined. Individualdeoxyribonucleosides were tested at concentrations of 20 μM, and thecombination of all four deoxyribonucleosides contained thymidine,deoxycytidine, deoxyadenosine and deoxyguanosine at 20 μM each.

TABLE 5 Effect of individual versus combined deoxyribonucleosides onUV-induced DNA damage DNA Strand Breaks Group (Breaks/47 MDa) Control(no UV) .0  UV + vehicle .325 UV + thymidine .13  UV + deoxycytidine.12  UV + deoxyadenosine .17  UV + deoxyguanosine .17  UV + dNsides (20μM each) .01 

As shown in Table 5, each of the individual deoxyribonucleosidesattenuated DNA damage; the combination of all four deoxyribonucleosideswas substantially more effective than any individual compound.

EXAMPLE 6

Sunscreen-induced Exacerbation of UV-induced Tumorigenesis and itsPrevention with Deoxyribonucleosides

A circular patch of commercial sunscreen (Coppertone SPF 8, whichincludes oxybenzone) was applied to the backs of TG.AC mice prior toexposure to 125 J/m² UV-B radiation.

Around the circular margin of the area that was covered with sunscreenduring irradiation, 8 papillomas per mouse were present at 10 days. 3tumors per mouse were observed in animals not treated with sunscreen. Inmice exposed to UV radiation after application of the same commercialsunscreen to which deoxyribonucleosides had been added, no papillomaswere elicited (Table 6).

TABLE 6 Deoxyribonucleosides attenuate tumorigenesis in in mice treatedwith UV plus sunscreen UV Ratiation 3 tumors/mouse Sunscreen + UVradiation 8 tumors/mouse Sunscreen containing dNsides + UV radiation 0tumors/mouse

Thus, at the margin of the applied patch of sunscreen where thesunscreen concentration diminishes in a rapid gradient toward zero,tumorigenic UV damage in fact appears to be enhanced rather than reducedby the sunscreen agents, leading to formation of more premalignantpapillomas than on skin not treated with sunscreen at all.

Addition of deoxyribonucleosides to a commercial sunscreen abrogated thedeleterious effect of the sunscreen on tumorigenesis.

EXAMPLE 7

Effect of Individual Deoxyribonucleosides Versus a Combination onUV-induced Tumorigenesis

Thirty v-Ha-ras TG.AC mice aged twelve weeks were shaved and subjectedto ultraviolet radiation, 1.25 kJ/m² on days 0, 6, 8 and 11, at a doserate of 12.5 W/m2.

Mice were divided into groups of five animals each and treated,beginning 30 minutes after irradiation, with:

1. Vehicle (propylene glycol)

2. dNsides—Deoxyribonucleosides (4 mg/ml each of deoxyadenosine,deoxycytidine, deoxyguanosine, and thymidine)

3. dC—Deoxycytidine (4 mg/ml)

4. dG—Deoxyguanosine (4 mg/ml)

5. dA—Deoxyadenosine (4 mg/ml)

6. dT—Thymidine (4 mg/ml)

Animals were observed for 8 weeks, during which time the development ofpapillomas was observed.

TABLE 7 Effect of individual versus combined deoxyribonucleosides onUV-induced tumorigenesis Papillomas/mouse at the end of 8 weeks Vehicle1.8 dNsides 0.0 dC 0.25 dG 1.0 dA 0.67 dT 1.6

As shown in Table 7, the mixture of all four deoxyribonucleosidesprovided the best activity in terms of prevention of tumor development.Deoxycytidine also provided protection; deoxyadenosine anddeoxyguanosine were less protective but nonetheless had activity.Thymidine did not have significant protective actions.

EXAMPLE 8

Nitric Oxide Causes and Enhances DNA Damage by aDeoxyribonucleoside-reversible Mechanism

Nitric oxide is a mediator of UV-induced erythema, and is also presentin inflammatory skin conditions. NO is mutagenic, and may exacerbateUV-induced DNA damage, in part by inhibiting ribonucleotide reductase,the enzyme responsible for conversion of ribonucleotides todeoxyribonucleotides.

Human melanocytes were exposed to 160 μM DETA NONOate (AlexisCorporation, Cat # 430-014-M005), a reagent which spontaneously producesnitric oxide when exposed to water. Cells were exposed to NO alone, orNO plus increasing doses of UV radiation (50 and 300 J/m²). Groups ofcells were also exposed to deoxyribonucleosides of the invention before,or before and after, exposure to NO or NO+UV radiation; control groupswere treated identically except for addition of deoxyribonucleosides.

DNA was extracted from cells and subjected to pulsed field gelelectrophoresis to determine the incidence of DNA strand breaks.

As shown in Table 8, NO alone produced a significant incidence of DNAstrand breaks, which were further increased by exposure to UV radiation.The deoxyribonucleosides of the invention strongly reduced the incidenceof DNA strand breaks caused by NO, as well as those caused by thecombination of NO plus UV radiation.

TABLE 8 Deoxyribonucleosides attenuate Nitric Oxide-induced DNA damageDNA Strand Breaks Groups (breaks/105 MDa) Control 0.00 NO 0.13 NO + UV50 J/m² 0.21 NO + UV 300 J/m² 0.51 NO + dNsides before and after UV 0.00NO + UV 50 J/m² + dNsides before/after UV 0.03 NO + UV 300 J/m² +dNsides before/after UV 0.03 NO + dNsides after UV 0.06 NO + UV 50J/m² + dNsides after UV 0.06 NO + UV 300 J/m² + dNsides after UV 0.03

EXAMPLE 9

Treatment of Existing Actinic Keratosis with TopicalDeoxyribonucleosides

Actinic keratosis (AK) is a form of intradermal neoplasia, asunlight-induced lesion which can progress to become squamous cellcarcinoma. A 46 year-old woman with very light skin presented with apersistent rose colored, rough-surfaced lesion 5×10 mm in size on herforehead. The lesion was diagnosed as an actinic keratosis by aboard-certified dermatologist, who prescribed topical 5-fluorouracil forits treatment. Since 5-fluororacil, a standard treatment for AK, canproduce pain and unsightly skin erosion which takes several weeks toheal and is also potentially genotoxic, the subject elected to first tryto treat the AK with topical deoxyribonucleosides (4 mg/ml each ofdeoxycytidine, deoxyadenosine, deoxyguanosine, and thymidine) in ahydrogel formulation, of which 0.1 ml was applied to the lesion twicedaily. Within 10 days, the AK on her forehead had begun to visiblyregress, and after three weeks, there was no trace of the AK, nor wasthere any scar or discomfort during or after treatment. 5-fluorouraciltreatment and other painful or invasive measures like excision orfreezing with liquid nitrogen were thus not needed. The AK has notrecurred after more than six months.

EXAMPLE 10

Formulation of a Composition of the Invention

A suitable clinical formulation of a composition of the inventioncomprises the following ingredients. The batch size is optionally scaledto any desired volume.

 1. 2′-Deoxycytidine Hydrochloride 0.4640 g  2. 2′-DeoxyadenosineMonohydrate 0.4290 g  3. 2′-Deoxyguanosine Monohydrate 0.4270 g  4.Thymidine 0.4000 g  5. Edetate Disodium Dihydrate, USP 0.0100 g  6.Benzethonium Chloride, USP 0.0250 g  7. Butylated Hydroxytoluene, NF0.0005 g  8. Carbomer 934P, NF 0.7200 g  9. Glycerin, USP 10.000 g 10.Alcohol, USP 5.000 g 11. Diethylene glycol monoethyl ether 10.000 g 12.NaOH, NF (1 N Solution) as needed for pH adjustment 13. HCl, NF (1 NSolution) as needed for pH adjustment 14. Purified Water, USP q.s. to100.00 g

Preparation:

1. Item 8 (Carbomer 934P) is dissolved in 35 g of Purified Water.

2. Item 5 is dissolved into the solution.

3. Into a separate container, Items 9, 10, 11 are mixed with 15 gPurified Water.

4. Items 1, 2, 3, 4, 6, and 7 are dissolved in the solution of step 3.

5. The solutions of step 2 and 4 are mixed together.

6. 8.85 g of Item 12 are added to the solution of step 5.

7. The resulting gel is blended for 15 minutes, and the pH is adjustedto 7.0+/−0.5 with Item 12 or 13 as needed.

8. The final weight of the batch is brought to 100 g with Purified Waterfollowed by gentle blending for 15 minutes.

The foregoing is intended as illustrative of the present invention butnot limiting. Numerous variations and modifications may be effectedwithout departing from the true spirit and scope of the invention.

What is claimed is:
 1. A method for reducing the chance of developingskin cancer in a mammal due to exposure to a mutagen comprisingadministering to the skin of said mammal a source of an individualdeoxyribonucleoside wherein said source is administered such that saiddeoxyribonucleoside is present on skin of said mammal during or afterexposure to said mutagen in an amount sufficient to reduce thedeleterious consequences of said exposure: wherein said source isselected from the group consisting of the deoxyribonucleoside in freeform or the corresponding deoxyribonucleotide.
 2. A method as in claim1, wherein said source is administered within 3 days after exposure to amutagen.
 3. A method as in claim 2 wherein said mutagen is selected fromthe group consisting of solar, ultraviolet, or ionizing radiation.
 4. Amethod as in claim 1 wherein said source is the freedeoxyribonucleoside.
 5. A method as in claim 1 wherein said source isthe corresponding deoxyribonucleotide.
 6. A method as in claim 1 whereinsaid source is administered in a vehicle at a concentration of from 1.0to 20 milligrams per milliliter.
 7. A method as in claim 4 wherein saiddeoxyribonucleoside is free deoxycytidine.
 8. A method as in claim 4wherein said deoxyribonucleoside is free deoxyadenosine.
 9. A method asin claim 4 wherein said deoxyribonucleoside is free deoxyguanosine. 10.A method as in claim 4 wherein said deoxyribonucleoside is freethymidine.
 11. A method as in claim 4, wherein said deoxyribonucleosideis administered in a vehicle containing from 0.1 to 10 milligrams permilliliter of said deoxyribonucleoside.
 12. A method as in claim 4wherein said at least one deoxyribonucleoside is administered in avehicle containing from 1.0 to 5 milligrams per milliliter of eachdeoxyribonucleoside.
 13. A method as in claim 1 wherein said skin canceris selected from the group comprising basal cell carcinoma, melanoma,and squamous cell carcinoma.
 14. A method of reducing the chance ofdeveloping skin cancer in a mammal due to exposure to solar orultraviolet radiation comprising topically administering a compositioncomprising a sunscreen agent and an energy scavenging agent: whereinsaid energy scavenging agent is selected from the group consisting of anindividual deoxyribonucleoside in free form or the correspondingribonucleoside, ribonucleotide, deoxyribonucleotide, or a correspondingacyl ribonucleoside.
 15. A method as in claim 14 wherein said sunscreenagent is selected from the group consisting of avobenzone (t-butyldimethoxydibenzoylmethane), oxybenzone (benzophenone-3), dioxybenzone(benzophenone-8), sulisobenzone (benzophenone-4;2-hydroxy-4-methoxybenzophenone-5-sulfonic acid), octocrylene(2-ethylhexyl-2-cyano-3 3-diphenylacrylate), octyl methoxycinnamate(2-ethyihexyl p-methoxycinnamate), octyl salicylate(2-ethylbexylsalicylate), homosalate (homomenthyl salicylate), trolaminesalicylate (triethanolamine salicylate), phenylbenzimidazole sulfonicacid, PABA (para-aminobenzoic acid), roxadimate (ethyl 4-bishydroxypropyl aminobenzoate), lisadimate (glyceryl PABA), Padimate 0(octyldimethyl PABA), menthyl anthranilate, or Parsol 1789 (butylmethoxydibenzoylmethane).
 16. A method as in claim 14 wherein saidenergy scavenging agent is administered in a vehicle containing saidagent at a concentration from 0.1 to 20 mg/ml.